Changes in the cytoskeleton and signalling pathways in differentiated human neuroblastoma cells following MPP+ exposure

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Beck, K.E., 2004. Changes in the cytoskeleton and signalling pathways in differentiated human neuroblastoma cells following MPP+ exposure. PhD, Nottingham Trent University.

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Abstract

l-methyl-4-phenyl-l,2,3,6 tetrahydropyridine (MPTP), via its active metabolite, 1- methyl-4-phenylpyridinium (MPP+), provides a Parkinsonian model for investigating (a) the mechanisms of cell death and (b) neuroprotective strategies. The majority of research uses either animal models or mitotic cell lines, exposed to MPTP/MPP+ over relatively short exposure times. In the work presented, the human neuroblastoma (SH-SY5Y) cell line was differentiated biochemically and morphologically to provide a viable, mature phenotype for extended time periods. Following a 7-day pre-differentiation regime, cells were exposed to cytotoxic and sub-cytotoxic concentrations of MPP+ over 72 h, and to sub-cytotoxic concentrations for up to 14 days. In the latter case cell viability is not significantly reduced but subtle biochemical changes occur that may replicate the neurodegenerative process in-vivo.

The effects of MPP+ and proteasomal inhibition on the cytoskeleton were investigated, in particular the neurofilament (NF) and microtubule (MT) systems. Results suggest that changes in post-translational modification/distribution of NFs represent down-stream markers of MPP+ toxicity in this system. Following 24 h exposure to cytotoxic, and 14 days exposure to sub-cytotoxic MPP+ concentrations, phosphorylated NF-H and NF-M levels increased, associated with an increase in the ratio of phosphorylated NF-M to NF-H. Immunocytochemical analyses of cells exposed to cytotoxic MPP+ levels revealed phosphorylated NF-H/NF-M as a discrete accumulation adjacent to and impinging on the nucleus. Similarly, in cells exposed to sub-cytotoxic MPP concentrations, NF-H/NF-M was located as a perinuclear halo. MPP+ exposure significantly increased levels of ubiquitinated proteins, suggestive of impaired proteasomal activity. Evidence was provided for proteasomal degradation of NFs and tubulin since they accumulated following proteasomal inhibition using a specific inhibitor. This project established a novel role for the transamidating/GTPase enzyme, tissue transglutaminase (tTG) in MPP+ toxicity. MPP+ increased tTG activity, despite reducing tTG protein levels, whilst inhibition of tTG activity exacerbated MPP+ toxicity.

The effects of specific MAPK pathway inhibitors on MPP+ toxicity revealed a dynamic balance between pathways. Protection conferred by MEK and p38 inhibitors was dependent on the level of MPP+ toxicity whilst inhibition of the JNK pathway attenuated MPP+ toxicity. The role of CDK-5 in this system was also investigated since it is predominantly expressed in post-mitotic neurones, is implicated in Alzheimer's disease and has recently been identified in brainstem and cortical Lewy bodies. Butyrolactone 1, a selective CDK-5 inhibitor significantly protected cells treated for up to 14 days with MPP+. This study is the first to show protection against MPP+ toxicity using a selective CDK-5 inhibitor (Butyrolactone I) in a human neuronal cell system.

Item Type:

Thesis

Creators:

Beck, K.E.

Date:

2004

ISBN:

9781369316681

Identifiers: